PROJECT SUMMARY Simultaneous replication and transcription of the same DNA template (replication-transcription conflicts) appear to be common in eukaryotic cells. Although these conflicts have long been studied as a potential source of DNA damage and, therefore, a threat to genome integrity, we lack a detailed molecular understanding of how the presence of transcribing RNA polymerases on DNA affects the movement of the replication machinery (replisome), and of the mechanism(s) by which these replication-transcription conflicts can ultimately give rise to DNA damage. The long-term goal of this project is to characterize the nature and outcomes of eukaryotic replication-transcription conflicts at the molecular level. The proposed work uses a recently developed quantitative method to assay the movement of the replisome genome-wide at high resolution, in combination with genome-wide interrogation of DNA double-strand break formation. Previous research has indicated that tRNA genes, which are transcribed by RNA polymerase III (RNAP3), represent sites of abundant DNA damage and strong, polar barriers to replication ? i.e. a tRNA gene in the head-on orientation relative to replisome movement leads to terminal arrest of a significantly higher fraction of replisomes than a tRNA gene oriented co-directionally with replication. In contrast, genes transcribed by RNA polymerase II (RNAP2) do not appear to induce significant replisome arrest but have previously been reported to be associated with DNA damage in an orientation-dependent fashion, especially in the context of impaired pre-mRNA processing. This study has two specific aims: (1) to determine the molecular basis of replisome arrest and DNA damage at tRNA genes, and (2) to establish which intermediate states of transcribing RNAP2 are responsible for impeding replisome movement and stimulating DNA damage. This work will be carried out in the budding yeast Saccharomyces cerevisiae: the small genome, rapid replication, and easy genetic manipulability of S. cerevisiae make this an ideal model in which to study the intersection of these fundamental biological processes. The core machineries responsible for both DNA replication and transcription are extremely highly conserved throughout eukaryotes. Therefore, the results of this work will provide molecular insights into transcription-mediated genome instability in humans, and be directly applicable to our understanding of both the etiology and progression of cancer.